Leukemia stem cells (LSC) comprise subpopulations of cells in acute or chronic leukemia that have acquired ?stem cell? properties including the ability to endure unlimited self-renewal, maintain aberrant clonal hematopoiesis and achieve quiescence upon exposure to chemotherapy or other bio-stressors thereby conferring resistance to antileukemia treatments. Currently available cell-cycle-dependent chemotherapy and other molecular targeting agents are unable to eliminate these LSCs. Thus new effective treatments to abrogate LSC are an unmet need. MicroRNAs (miRNAs) are short non-coding RNAs that regulate levels of multiple target proteins, thereby controlling a wide array of cellular programs. Among miRNAs that are deregulated in leukemia, higher expression of miR-126-3p (miR-126) is associated with LSC-gene expression signatures and poor outcome. Furthermore, higher levels of miR-126 controls quiescence both in normal hematopoietic stem cells (HSC) and LSC, but while attenuated miR-126 activity increases HSC hematopoietic output, it drives LSC to exhaustion. The central hypothesis of this proposal is that miR-126 is critical for the homeostasis of LSC and mediates LSC therapy resistance, thus represents a promising LSC-directed therapeutic target. The major objective of this application is to understand how miR-126 expression is aberrantly regulated in LSC and to develop an effective therapeutic approach to inhibit miR-126 in LSC, while sparing normal hematopoiesis. As a proof-of-principle, we will focus on targeting miR-126 in acute myeloid (AML) and chronic myeloid leukemia (CML), but similar principles could be expanded to other types of leukemia. We propose the following specific aims (SA): SA1. To dissect and overcome the molecular mechanisms of therapy resistance mediated by a newly discovered SPRED1/miR-126 autoregulatory loop in LSC. We will test that a tyrosine kinase (TK)-dependent SPRED1/miR-126 autoregulatory loop is operative in AML and CML, which mediate miR-126-dependent mechanisms of resistance to tyrosine kinase inhibitors (TKI). We will 1) assess the activity of SPRED1/miR-126 autoregulatory loop in distinct subtypes of AML; 2) define TK-dependent SPRED1 phosphorylation sites; 3) create leukemia mouse models to dissect the interplay of SPRED1/miR-126 autoregulatory loop with aberrantly active TK. SA2. To define the role of miR- 126 in maintaining a LSC niche within the bone marrow microenvironment. We will develop genetically engineered AML and CML models with conditional miR-126 deletion in LSC and endothelial cells (EC). We will determine 1) the contribution of miR-126 produced by LSC; 2) the contribution of miR-126 in the EC compartment; 3) whether deletion of miR-126 could enhance treatment-mediated elimination of LSC. SA3. To develop and optimize a synthetic inhibitor that targets miR-126 in LSC and the LSC niche. We will perform PK and PD analyses and preclinical studies to define the active dose/schedule of an antimiR-126 conjugated with CpG-oligodeoxynucleotide (ODN) for optimal targeted cell delivery.